CN102272315A - Improvement of enzymatic hydrolysis of pretreated lignocellulose-containing material with dissolved air flotation sludge - Google Patents
Improvement of enzymatic hydrolysis of pretreated lignocellulose-containing material with dissolved air flotation sludge Download PDFInfo
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- CN102272315A CN102272315A CN2009801534022A CN200980153402A CN102272315A CN 102272315 A CN102272315 A CN 102272315A CN 2009801534022 A CN2009801534022 A CN 2009801534022A CN 200980153402 A CN200980153402 A CN 200980153402A CN 102272315 A CN102272315 A CN 102272315A
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- lignocellulose
- containing materials
- air flotation
- dissolved air
- enzyme
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/02—Monosaccharides
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- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
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- C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
- C12P7/06—Ethanol, i.e. non-beverage
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Abstract
A method for producing a fermentation product from a lignocellulose-containing material comprises pretreating the lignocellulose-containing material; introducing treated dissolved air flotation sludge to the pretreated lignocellulose-containing material; exposing the pretreated lignocellulose-containing material to a hydrolyzing enzyme; and fermenting with a fermenting organism to produce a fermentation product.
Description
Technical field
Disclose the method that is used for producing tunning, more specifically, disclose by increasing from the method for the efficient of this material production tunning with dissolved air flotation sludge conditioning lignocellulose-containing materials from lignocellulose-containing materials.
Background technology
Lignocellulose-containing materials, or claim biomass, can be used for producing fermentable carbohydrate, it can be then used in and produce tunning such as reproducible (renewable) fuel and chemical.Lignocellulose-containing materials has cellulosic fibre and is wrapped in complex construction in xylogen and the hemicellulose sheath.Produce tunning from lignocellulose-containing materials and comprise pre-treatment, hydrolysis and the described lignocellulose-containing materials that ferments.
Lignocellulose-containing materials is converted into physics, biology, chemistry and/or the enzyme that reproducible fuel and chemical usually relate to biomass to be handled.Particularly, enzyme is a D-glucose with cellulose hydrolysis, and it is simple fermentable sugar.Because the high lignin content in many lignocellulose-containing materials needs the enzyme of high dosage to come with the high yield degraded cellulose.This may be because xylogen and lignin derivative suppress lytic enzyme.Above-mentioned inhibition can be carried out at least in two ways: xylogen or lignin derivative are preferentially in conjunction with described enzyme, thereby stop this enzyme to combine or hydrocellulose with Mierocrystalline cellulose, and/or xylogen or lignin derivative cover a cellulosic part, thereby reduce enzyme and cellulosic approaching.As a result, when the biomass of processing high lignin content, can be used for the enzyme of degraded cellulose may be less, because xylogen and derivative thereof can be removed described enzyme or block its activity.Even for being used for the enzyme of degraded cellulose, usually the available enzyme also can't contact with Mierocrystalline cellulose, because xylogen covers Mierocrystalline cellulose.Therefore, reduced the validity of the technology of digest cellulose.In addition, the cost height of enzyme.Therefore, when the required enzyme amount of degraded cellulose was higher, tooling cost is high, and was inadvisable economically.
The minimizing that obtains the required enzyme amount of gratifying sugar yield can have remarkable influence to process economics.Therefore, improvement enzyme service efficiency is the main demand in the bio-conversion process.Think that several factors influences cellulosic enzymic hydrolysis.These factors comprise content of lignin, hemicellulose level, acetyl content, cellulosic surface-area and Mierocrystalline cellulose crystallinity.Usually it should be understood that the xylogen that is present in the complex substrate has detrimental action to enzymic hydrolysis.
Still difficult definite xylogen and the accurate effect of lignin derivative in limiting hydrolysis.Yet the effect of known removal xylogen and derivative thereof increases cellulosic hydrolysis and increases the productive rate of fermentable sugar.This effect may have been opened the more plain surface-area of multifilament and attacked for enzyme, and may reduce the enzyme amount of non-specific adsorption on the ligno-cellulose substrate.Perhaps, the compound that is used to remove xylogen and derivative thereof can make Mierocrystalline cellulose more be subject to enzyme liberating, therefore reduces required enzyme amount and increases the ethanol yield of biomass to the ethanol.
The accompanying drawing summary
Fig. 1 shows the figure of dissolved air flotation sludge to the effect of the glucose yield of the PCS hydrolysis gained of the washing of hanging oneself.
Fig. 2 shows the figure of dissolved air flotation sludge to the effect of the conversion of glucose per-cent of the PCS hydrolysis gained of the washing of hanging oneself.
Summary of the invention
Disclose in the presence of treated dissolved air flotation sludge to come from the method for this material production tunning by pre-treatment and/or hydrolysis lignocellulose-containing materials.
Also disclose the method that is used for producing tunning, comprised the described lignocellulose-containing materials of pre-treatment from lignocellulose-containing materials; Treated dissolved air flotation sludge is introduced through pretreated lignocellulose-containing materials; Be exposed to lytic enzyme with described through pretreated lignocellulose-containing materials; With with fermenting organism fermentation to produce tunning.In one aspect, can before the lytic enzyme that described lignocellulose-containing materials is exposed to significant quantity, described treated dissolved air flotation sludge be introduced this lignocellulose-containing materials.
The method of the enzymic hydrolysis that is used to strengthen lignocellulose-containing materials is disclosed in addition.Aforesaid method comprises to be introduced the dissolved air flotation sludge of effective xylogen blocking-up amount described lignocellulose-containing materials and described lignocellulose-containing materials is exposed to lytic enzyme.
Detailed Description Of The Invention
Disclose improved and more effective by using treated dissolved air flotation sludge to contain the method for the biomass of xylogen with enzymic hydrolysis as xylogen blocking-up thing.
Xylogen is to pass through the dehydrogenation polymerization deutero-phenol polymer of lubanol and/or sinapyl alcohol, and sees in the cell walls of various plants.Term " xylogen " refers to the complete structure of lignin polymers and destroys any derived fragment or the compound of the described complete polymkeric substance obtain by lignin structure as used in this article, comprises (condensed) xylogen and the insolubility precipitation xylogen of solubility lignin derivative, condensation.Think that various lignin derivatives are different in the interaction of itself and treated dissolved air flotation sludge.For example, think that the xylogen of insolubility precipitation xylogen and condensation has from the ability of the treated dissolved air flotation sludge of aqueous solution absorption.
Term " biomass slurry " refers to take place the water-based biological material of enzymic hydrolysis as used in this article.Biomass slurry produces by biomass examples such as maize straw, bagasse etc. are mixed with water, damping fluid and other material previously treateds.But preprocessing biomass before hydrolysis.
Term " xylogen blocking-up (lignin blocking) " means the deleterious effect that reduces or eliminates xylogen in the technology that with Wood Adhesives from Biomass is tunning as used in this article.In addition, term " effectively xylogen blocking-up amount " means any amount that can be used for promoting the xylogen blocking-up as used in this article.
In one embodiment, present method has been utilized treated dissolved air flotation sludge.Be not limited to any concrete theory, think that treated dissolved air flotation sludge can be preferentially easier in xylogen than Mierocrystalline cellulose.Available treated dissolved air flotation sludge conditioning biomass slurry is for example by directly introducing treated dissolved air flotation sludge through pretreated biomass slurry.Therefore think that treated dissolved air flotation sludge is preferentially combining in pretreated slurry with xylogen, thereby covered the xylogen that is deposited in cellulose surface, hinder combining of sedimentary xylogen and lytic enzyme.Cellulolytic enzyme hydrocellulose more effectively then.If dissolved air flotation sludge conditioning that need not be treated contains the biomass slurry of xylogen, but the part of xylogen cellulose-binding lytic enzyme makes it can't hydrocellulose, maybe can cover a Mierocrystalline cellulose part, and it is approaching to make it be hydrolyzed enzyme.
Be not limited to any concrete theory, think that xylogen does in order to inhibitory enzyme in many ways to cellulosic hydrolysis in the biomass.Xylogen restriction cellulolytic enzyme and hemicellulose lytic enzyme can be converted into Mierocrystalline cellulose and hemicellulose the degree of monose.The focus of many research activitiess is to understand the characteristic of xylogen in the cell walls, and exploitation is with its pretreatment technology of effectively removing.Understanding by xylogen inhibitory enzyme activity pattern can reduce traditionally the deleterious effect that the xylogen composition by biomass causes.As will be described in further detail below, can before hydrolysis lignocellulose-containing materials or biomass, carry out pre-treatment to it.For example, pre-treatment can be adopted the form of steam pre-treatment, alkaline pre-treatment, acid pre-treatment or its certain combination.Steam pre-treatment is physically opened the structure of biomass, promptly interrupts the key that connects xylogen, Mierocrystalline cellulose and hemicellulose at least in part.The alkalescence pre-treatment generally includes with alkaline matter such as ammonia treatment biomass.The alkalescence pre-treatment chemically changes biomass.For the xylogen composition of biomass, think to produce lignin derivative and little phenols fragment thus by alkaline pre-treatment lignin degrading at least in part that it can influence performance and the zymic growth and the fermentation capacity of enzyme unfriendly.Acid pre-treatment also chemically changes the xylogen composition of biomass, generates the lignin derivative of the xylogen that comprises condensation thus, and it is deposited in the cellulosic fibre surface.The xylogen of described condensation is by covering the surperficial inhibitory enzyme contact Mierocrystalline cellulose of cellulosic fibre.But other lignin derivatives that generate in acid preprocessing process comprise the little phenol fragment and the compound of containing of inhibitory enzyme function.
Think that also with treated dissolved air flotation sludge conditioning biomass slurry to small part be by in conjunction with xylogen, thereby reduce and/or suppress xylogen the unproductive absorption of cellulolytic enzyme is worked.In addition, think that dissolved air flotation sludge advantageously works as the tensio-active agent of described enzyme.Think that tensio-active agent improves the accessibility (accessibility) of substrate, improves enzyme stability, and reduces unproductive xylogen combination.Think that these advantages are attributable to described tensio-active agent enzyme is maintained in the solution, therefore make enzyme potentially, stabilized enzyme, and the useful life (productive life) that has prolonged enzyme away from xylogen.Therefore, with treated dissolved air flotation sludge conditioning biomass slurry by suppressing the xylogen desmoenzyme and improving enzymic hydrolysis and improve to containing the processing of xylogen substrate.Treated dissolved air flotation sludge can reduce the enzyme heap(ed) capacity and/or improve enzyme performance, because described enzyme is not subjected to the disadvantageous effect of xylogen so, therefore more enzyme still can be used for more effectively hydrolyzing biomass slurry.In addition, the useful life of enzyme obtains prolonging by the Action of Surfactant of described dissolved air flotation sludge.
Present method reduces the enzyme heap(ed) capacity in the hydrolysis of the biomass slurry that contains xylogen.By being added into biomass slurry, treated dissolved air flotation sludge significantly reduced the enzyme amount that is hydrolyzed required.Reduce the total cost that the enzyme heap(ed) capacity has reduced Wood Adhesives from Biomass technology.
According to an embodiment, described method uses treated dissolved air flotation sludge to strengthen cellulosic enzymic hydrolysis.This method comprises the biomass slurry that contains xylogen with treated dissolved air flotation sludge conditioning obtaining the having treated biomass slurry of the lignin component of being blocked, and treated biomass slurry is exposed to the step of the lytic enzyme of significant quantity.Described dissolved air flotation sludge can be in preprocessing process or afterwards, or directly makes an addition to described biomass slurry before the hydrolysis or in the process.Preferably before adding cellulolytic enzyme and fermenting organism, described dissolved air flotation sludge is added into biomass slurry.
Dissolved air flotation sludge
The dissolved air flotation is to be widely used in the water technology of industry as food-processing and oil refining.The dissolved air flotation makes the waste water clarification by removing suspended matter such as oil or solid substance.A large amount of light solid substances and hydrophobic material are removed from the dissolved air flotation as sludge as fat, oil and grease (grease).This sludge can be used for the described technology of the application.
The principle of dissolved air floatation process under atmospheric pressure discharges described air then for air is dissolved in the waste water in flotation tank or basin under pressure.The air that discharges forms small bubble, and it invests suspended matter and causes described suspended matter floating to fluid surface, can be removed by skimmer device this its then.
The dissolved air flotation is widely used for handling the trade effluent effluent from refinery, petroleum chemistry and chemical plant, gas plant and similar industrial facility very much.It also is used to handle the waste water from agriculture technology.
Agricultural effluent is handled and relate to the waste water that processing produces in the rural activity process.Agricultural technology can generate waste water, and it comprises, and animal waste, silage slurries (silage liquor), outflow and excessive sterilant (pesticide run off and surpluses), milking parlour refuse comprise milk, butcher refuse, plant washing water and fire water.The waste water of agricultural technology contains following component usually: high organic components, high solid concentration, high nitrate and phosphorus composition, microbiotic, synthetic hormone, the usually parasite and the ovum thereof of high density, and the gemma of various bacteria.It also can contain washes (wash-down water) and the cleaning and the sterilization chemical of large volume.
Be used for dissolved air flotation sludge of the present invention and can comprise refuse from the wastewater treatment that relates to agriculture technology.More specifically, it can comprise from the refuse that relates to the wastewater treatment of butchering technology.The example of available dissolved air flotation sludge is for butchering the dissolved air flotation sludge of wastewater treatment in the technology from pig.Using dissolved air flotation sludge is favourable economically to improve enzymic hydrolysis.It has increased the generation recirculation simultaneously of tunning and has been used to natural refuse from agriculture technology, and it does not need the high treatment process of use cost to handle and/or abandon.In addition, think that adding dissolved air flotation sludge can reduce or alleviate with the hydrolysis that improves biomass slurry nitrogen is made an addition to the demand that biomass slurry is used for zymotechnique.Usually, after hydrolysis, nitrogen is added into described biomass slurry to improve zymotechnique by the situation of improving fermenting organism.Yet the nitrogen that dissolved air flotation sludge contains capacity makes an addition to biomass slurry to improve the demand of zymotechnique to reduce or to alleviate after hydrolysis with more nitrogen.
Contained dissolved air flotation sludge can be before introducing biomass slurry treated or processing to kill the living organism in the sludge.If do not kill living organism, thereby its simple carbohydrate that may consume from hydrolysis process reduces fermentation available sugar amount.Handle or process the combination that can comprise enzyme method, by the use of thermal means, mechanical means, chemical process or method.Can before being introduced biomass slurry, dissolved air flotation sludge carry out autoclaving to it.For example, described dissolved air flotation sludge can be 121 ℃ of autoclavings 20 minutes.
Think and at first use treated dissolved air flotation sludge conditioning biomass slurry, add cellulolytic enzyme then and make cellulose conversion have the highest efficient.With treated dissolved air flotation sludge conditioning biomass slurry also can with cellulolytic enzyme is added into biomass slurry and carries out simultaneously.
Be not limited to any concrete theory, think treated dissolved air flotation sludge to the non-specific binding of xylogen reduced enzyme to the unproductive combination on xylogen surface or since with the inhibition of the interaction partners enzymic activity of xylogen.Therefore, use treated dissolved air flotation sludge to help promoting being used to obtaining the decline of the enzyme heap(ed) capacity of identical target percentage conversion being used for technology that ligno-cellulose transforms.
Lignocellulose-containing materials
" ligno-cellulose " or " lignocellulose-containing materials " means the material of mainly being made up of Mierocrystalline cellulose, hemicellulose and xylogen.Above-mentioned materials usually is known as " biomass ".
Biomass have cellulosic fibre and are wrapped in complex construction in xylogen and the hemicellulose sheath.The structure of biomass makes it not be subject to enzymic hydrolysis.In order to strengthen enzymic hydrolysis, must preprocessing biomass, for example by carrying out acid hydrolysis to break the sealing of xylogen under enough pressure and temperature conditions, saccharification is also dissolved hemicellulose, and destroys cellulosic crystalline structure.Then can be with the Mierocrystalline cellulose enzymic hydrolysis, for example handle by cellulolytic enzyme, glycopolymers is converted into to ferment is required tunning such as the fermentable carbohydrate of alcoholic acid.Also can use the hemicellulose lytic enzyme to handle hydrolysis any residual hemicellulose in pretreated biomass.
Biomass can be any material that contains ligno-cellulose.In a preferred embodiment, biomass contain at least about 30wt.%, preferably at least about 50wt.%, and more preferably at least about 70wt.%, even more preferably at least about the 90wt.% ligno-cellulose.It should be understood that described biomass also can comprise other compositions such as protein material, starch and the carbohydrate carbohydrate as fermenting and maybe can not fermenting, or its mixture.
Biomass see leaf, branch and wooden (wood) of for example stem, leaf, shell/pod (hull), shell/skin/pod/bud (husk) and cob or the tree of plant usually.Biomass include but are not limited to draft material, agricultural residue, forestry resistates, municipal solid waste, waste paper and paper pulp and paper mill resistates.It should be understood that biomass can be the form that contains the Plant cell wall material of xylogen, Mierocrystalline cellulose and hemicellulose in blended matrix.
The example of the biomass that other are suitable comprises that zein fiber, rice straw, pine, wood shavings (wood chip), bagasse, paper and paper pulp processing refuse, maize straw, corn cob, hardwood such as poplar and birch, cork, grain straw such as straw, rice straw, switchgrass, awns belong to (Miscanthus), rice husk, municipal solid waste (MSW), industrial organic waste, office with paper or its mixture.
In a preferred embodiment, described biomass are selected from one or more in maize straw, corn cob, zein fiber, straw, rice straw, switchgrass and the bagasse.
Pre-treatment
Described biomass can be carried out pre-treatment in any suitable manner.According to the present invention, pre-treatment can comprise introduces described biomass with dissolved air flotation sludge.
Pre-treatment was carried out before hydrolysis or fermentation.Pretreated target is to separate or release Mierocrystalline cellulose, hemicellulose and xylogen, thereby improves hydrolysis rate or effectiveness.Pretreatment process (comprising wet oxidation and alkaline pre-treatment) target xylogen discharges, and diluted acid is handled and discharge from hydrolysis (auto-hydrolysis) target hemicellulose.Vapor explosion is the pretreatment process that the target Mierocrystalline cellulose discharges.
Pre-treatment step can comprise the step that wherein dissolved air flotation sludge is added into biomass.As previously shown, when adding dissolved air flotation sludge, biomass are generally the form of biomass slurry.If dissolved air flotation sludge is added into biomass slurry in preprocessing process, it is conventional that the rest part of pretreatment technology still keeps.Yet, perhaps can with dissolved air flotation sludge after pre-treatment and before the hydrolysis, or in hydrolysing step, add, thereby make that pre-treatment step is to use the conventional pre-treatment step of technology well-known in the art.
Dissolved air flotation sludge can add with the amount of about 1 to 40%w/w dissolved air flotation sludge/lignocellulose-containing materials.Preferably, the amount that they can about 5 to 20%w/w dissolved air flotation sludge/lignocellulose-containing materials is added.In a preferred embodiment, the biomass pre-treatment betides in the water paste.Biomass can be with the amount of about 10-80wt.% in preprocessing process, and preferably the amount of the amount of about 20-70wt.%, particularly about 30-60wt.% exists, and the amount of 50wt.% exists according to appointment.
Chemistry, machinery and/or Biological Pretreatment
Before hydrolysis or in the process, described biomass can be through chemistry, machinery, Biological Pretreatment, or its arbitrary combination.
Preferred described chemistry, machinery or Biological Pretreatment were implemented before hydrolysis.Perhaps, described chemistry, machinery or Biological Pretreatment can be implemented simultaneously with hydrolysis, as with add one or more cellulolytic enzymes or other enzymic activitys simultaneously, to discharge for example fermentable carbohydrate such as glucose or maltose.
In one embodiment, can wash in another way or detoxify through pretreated biomass.Yet, washing or detoxifcation and nonessential.In a preferred embodiment, through pretreated biomass through the washing or the detoxifcation.
Chemical Pretreatment
Phrase " Chemical Pretreatment " refers to promote any Chemical Pretreatment of the separation or the release of Mierocrystalline cellulose, hemicellulose or xylogen.The example of suitable chemically pretreating process comprises with for example diluted acid, lime, alkali, organic solvent, ammonia, sulfurous gas or carbonic acid gas to be handled.In addition, the aquathermolysis (hydrothermolysis) of wet oxidation and control pH also is considered as Chemical Pretreatment.
In a preferred embodiment, described Chemical Pretreatment is acid treatment, more preferably, for successive diluted acid or weak acid (mild acid) processing, for example, use sulfuric acid, or use other organic acids, as acetate, citric acid, tartrate, succsinic acid, hydrochloric acid or its mixture process.Also can use other acid.The pH that the weak acid processing means processing is at about pH 1-5, preferably in the scope of about pH 1-3.In a specific embodiments, described acid concentration arrives in the scope of 2.0wt% acid 0.1, and is preferably sulfuric acid.This acid can contact with described biomass, and mixture can be maintained at about 160-220 ℃, the temperature in 165-195 ℃ of scope according to appointment, the treatment time be several minutes to the several seconds, for example, 1-60 minute, as 2-30 minute or 3-12 minute.Can add strong acid (as sulfuric acid) and remove hemicellulose.The interpolation of strong acid has strengthened cellulosic digestibility.
Also contain other chemical pretreatment techniques according to the present invention.The plain solvent treatment of display fibers is a glucose with about 90% cellulose conversion.Also shown and when ligno-cellulose is destructurized, greatly strengthened enzymic hydrolysis.Alkali, H
2O
2, ozone, organic solvent (uses the Lewis acid in the aqueous alcohol, FeCl
3, (Al)
2SO
4), glycerine, two
Alkane, phenol or ethylene glycol belong to known destruction cellulosic structure and promote the solvent of hydrolysis (Mosier etc., 2005, Bioresource Technology 96:673-686).
Use alkali has also been contained in the present invention, NaOH for example, Na
2CO
3Alkali electroless pre-treatment with ammonia etc.Use the pretreatment process of ammonia to be described in for example WO 2006/110891, WO 2006/110899, WO2006/110900, WO 2006/110901, it incorporates this paper into by carrying stating.
Wet oxidation techniques relates to the use oxygenant, as, based on oxygenant of sulphite etc.The example of solvent pre-treatment comprises the processing with DMSO (methyl-sulphoxide) etc.Chemical Pretreatment was carried out 1 to 60 minute usually, as 5 to 30 minutes, carried out the short or long time but can be dependent on pending pretreated material.
The case description of other appropriate pretreatment methods is in Schell etc., 2003, Appl.Biochem and Biotechn.105-108 volume: 69-85 and Mosier etc., 2005, Bioresource Technology 96:673-686, and U. S. application discloses No. 2002/0164730, its each all incorporate this paper into by carrying stating.
Mechanical pretreatment
Phrase " mechanical pretreatment " refers to pre-treatment any machinery or physics, and it promotes authigenic material to separate or discharges Mierocrystalline cellulose, hemicellulose or xylogen.For example, mechanical pretreatment comprise polytypely grind, irradiation, decatize/vapor explosion (steam explosion), and aquathermolysis.
Mechanical pretreatment comprises pulverizing, and promptly machinery reduces size.Pulverizing comprises dry grinding, wet-milling and vibratory milling (vibratory ball milling).Mechanical pretreatment can relate to high pressure and/or high temperature (vapor explosion)." high pressure " means pressure about 300 to 600psi, and preferred 400 in the scope of 500psi, for example about 450psi.High temperature means temperature at about 100 to 300 ℃, in preferred about 140 to 235 ℃ scope.In a preferred embodiment, mechanical pretreatment is the vapor gun hydrolyzer system of batch process, and it uses high pressure and high temperature as defined above.Also can use Sunds Hydrolyzer (can obtain) by Sunds Defibrator AB (Sweden) for this reason.
The chemistry and the mechanical pretreatment of combination
In a preferred embodiment, biomass chemistry and two kinds of pre-treatment of machinery have been carried out.For example, described pre-treatment step can relate to diluted acid or weak acid processing and high temperature and/or autoclaving.Described chemistry and mechanical pretreatment can be as required order or carry out simultaneously.
Therefore, in a preferred embodiment, biomass are carried out chemistry and mechanical pretreatment with promotion the separating or release of Mierocrystalline cellulose, hemicellulose or xylogen.
In a preferred embodiment, described pre-treatment is carried out as diluted acid or weak acid pre-treatment step.In a further preferred embodiment, pre-treatment is carried out as ammonia fiber blast (fiber explosion) step (or AFEX pre-treatment step).
Biological Pretreatment
Phrase " Biological Pretreatment " refers to promote that authigenic material separates or discharge any Biological Pretreatment of Mierocrystalline cellulose, hemicellulose or xylogen.The Biological Pretreatment technology can relate to the microorganism of using dissolved lignin.Referring to, for example, Hsu, T.-A., 1996, Pretreatment of biomass, in Handbook on Bioethanol:Production and Utilization, Wyman, C.E. compiles, Taylor ﹠amp; Francis, Washington, DC, 179-212; Ghosh, P. and Singh, A., 1993, Physicochemical and biological treatments for enzymatic/microbial conversion of lignocellulosic biomass, Adv.Appl.Microbiol.39:295-333; McMillan, J.D., 1994, Pretreating lignocellulosic biomass:a review is in Enzymatic Conversion of Biomass for Fuels Production, Himmel, M.E., Baker, J.O. and Overend, R.P. compile, ACS Symposium Series 566, American Chemical Society, Washington, DC, the 15th chapter; Gong, C.S., Cao, N.J., Du, J. and Tsao, G.T., 1999, Ethanol production from renewable resources, in Advances in Biochemical Engineering/Biotechnology, Scheper, T. compiles, Springer-Verlag Berlin Heidelberg, Germany, 65:207-241; Olsson, L. and Hahn-Hagerdal, B., 1996, Fermentation of lignocellulosic hydrolysates for ethanol production, Enz.Microb.Tech.18:312-331; And Vallander, L. and Eriksson, K.-E.L., 1990, Production of ethanol from lignocellulosic materials:State of the art, Adv.Biochem.Eng./Biotechnol.42:63-95.
Hydrolysis
In fermentation before, can be with its hydrolysis so that Mierocrystalline cellulose and hemicellulose be degraded to fermentable carbohydrate through pretreated biomass (being preferably the form of biomass slurry).In a preferred embodiment, through pretreated material the fermentation before through hydrolysis, the preferred enzyme hydrolysis.
Dried solids content in the hydrolytic process can be about 5-50wt.%, preferably about 10-40wt.%, the preferably scope of about 20-30wt.%.In a preferred embodiment, hydrolysis can be used as the fed-batch process implementing, wherein will enrich the hydrating solution that for example contains enzyme gradually through pretreated biomass (that is substrate).
In a preferred embodiment, hydrolysis is implemented by enzyme.According to the present invention, can make up hydrolysis by one or more cellulolytic enzymes such as cellulase or hemicellulase or its through pretreated biomass slurry.
In a preferred embodiment, hydrolysis is to use following cellulolytic enzyme prepared product to implement, and it comprises one or more polypeptide with cellulolytic enhancing activity.In a preferred embodiment, the polypeptide with cellulolytic enhancing activity derives from the GH61A of family.The case description of suitable and preferred cellulolytic enzyme prepared product and the polypeptide with cellulolytic enhancing activity is in following " cellulolytic enzyme " part and " Mierocrystalline cellulose decomposes the enhancing polypeptide " part.
Because biomass can contain the component except xylogen, Mierocrystalline cellulose and hemicellulose, hydrolysis and/or fermentation can be implemented in the presence of other enzymic activitys such as protease activity, amylase activity, sugar generation enzymic activity and esterase activity such as lipase activity.
Enzymic hydrolysis is preferably implemented under the condition that those skilled in the art can easily determine in suitable aqueous environments.In a preferred embodiment, hydrolysis is preferably under the optimal conditions and implements suitable to described enzyme.
Suitable process time, temperature and pH condition can easily be determined by those skilled in the art.Preferably, hydrolysis is at 25 to 70 ℃, and preferred 40 to 60 ℃, particularly about 50 ℃ temperature is implemented.Hydrolysis is preferably at pH 3-8, preferred pH 4-6 scope, the pH enforcement of particularly about pH 5.In addition, hydrolysis was implemented 12 to 192 hours usually, and preferred 16 to 72 hours, more preferably 24 to 48 hours.
Fermentation
Hanging oneself fermentable carbohydrate of biomass of pre-treatment and/or hydrolysis can be by the fermentation of one or more fermenting organisms, and described fermenting organism can directly or indirectly be fermented into desired fermentation product with carbohydrate such as glucose, wood sugar, seminose and semi-lactosi.Fermentation condition depends on desired fermentation product and fermenting organism, and can be determined by a those of ordinary skill of this area.
Particularly under the situation of ethanol fermentation, fermentation can be carried out 1-48 hour, preferred 1-24 hour.In one embodiment, fermentation is at about 20-40 ℃, and preferably about 26-34 ℃, particularly about 32 ℃ temperature is carried out.In one embodiment, pH is greater than 5.In another embodiment, pH is about pH 3-7, preferred 4-6.Yet for example, some fermentation using bacteria biology has higher optimum leavening temperature.Therefore, in one embodiment, fermentation is carried out as 50-60 ℃ temperature at about 40-60 ℃.Those skilled in the art can easily determine suitable fermentation condition.
Fermentation can be in batches, implement in fed-batch or the flow reactor.Fed-batch fermentation can be constant volume (fixed volume) or transfiguration (variable volume) fed-batch.In one embodiment, use fed-batch fermentation.The volume of fed-batch fermentation and rate dependent in, for example identity of fermenting organism, fermentable carbohydrate (identity) and concentration and required tunning.Above-mentioned fermentation rate and volume can easily be determined by those of ordinary skills.
SSF, HHF and SHF
Hydrolysis and fermentation can be used as hydrolysis simultaneously and fermentation step (SSF) carries out.Usually this means combination/hydrolysis simultaneously and fermentation suitable to described fermenting organism, and preferably optimal conditions (for example, temperature and/or pH) are implemented down.
Hydrolysing step and fermentation step can be used as mixed hydrolysis and fermentation (HHF) is implemented.HHF begins with independent partial hydrolysis step usually, and finishes with while hydrolysis and fermentation step.Independent partial hydrolysis step is an enzyme process saccharification of cellulose step, and usually suitable to described lytic enzyme, preferred optimal conditions (for example at comparatively high temps) are implemented down.Hydrolysis and fermentation step are being implemented down fermenting organism appropriate condition (usually than the lower temperature of described independent hydrolysing step) usually in the time of follow-up.
Hydrolysis and fermentation step also can be used as independent hydrolysis and fermentation step carries out, and wherein said hydrolysis was finished before starting fermentation.This is often referred to as " SHF ".
Reclaim
After fermentation, can be randomly in the substratum that ferment separate fermentation product in any suitable manner.For example, retortable fermention medium to be extracting tunning, or extracts tunning by micro-filtration or membrane filtration technique in the substratum that can ferment certainly.Perhaps, can pass through stripping (stripping) and reclaim tunning.Recovery method is well-known in this area.
Tunning
The present invention can be used for producing any tunning.Preferred tunning comprises alcohols (for example, ethanol, methyl alcohol, butanols); Organic acid (for example, citric acid, acetate, methylene-succinic acid, lactic acid, glyconic acid); Ketone (for example, acetone); Amino acid (for example, L-glutamic acid); Gas (for example, H
2And CO
2); Microbiotic (for example, penicillin and tsiklomitsin); Enzyme; VITAMIN (for example, riboflavin, B
12, β-Hu Luobusu); And hormone.
Other products comprise consumption alcohols industrial product, for example, and beer and grape wine; The dairy products industry product, for example, the milk-product of fermentation; Leather industry product and tobacco industry product.In a preferred embodiment, described tunning is alcohol, particularly ethanol.The tunning (as ethanol) that obtains according to the present invention can be preferably used as fuel alcohol/ethanol.Yet for ethanol, it also can be used as drinking alcohol.
Enzyme
In the context of method of the present invention or technology,, will be understood that also enzyme and other compounds use with significant quantity even do not mention especially.Can use one or more enzymes.
The employed phrase of this paper " cellulolytic activity " is understood to include the have cellobiohydrolase activity enzyme of (EC 3.2.1.91), for example, cellobiohydrolase I and cellobiohydrolase II, and enzyme with endoglucanase activity (EC 3.2.1.4) and beta-glucosidase activity (EC3.2.1.21).
In a preferred embodiment, described cellulolytic activity can be the form of the enzyme prepared product of originated from fungus, as bacterial strain from Trichoderma (Trichoderma), and the bacterial strain of preferred Trichodermareesei (Trichoderma reesei); The bacterial strain of Humicola (Humicola) is as the bacterial strain of special humicola lanuginosa (Humicola insolens); Or the bacterial strain of Chrysosporium (Chrysosporium), the bacterial strain of preferred Chrysosporium lucknowense.
Described cellulolytic enzyme prepared product can contain one or more following activity: enzyme, seminase (hemienzyme), cellulolytic enzyme enhanced activity, beta-glucosidase activity, endoglucanase, cellobiohydrolase or xylose isomerase.
Described enzyme can be as the composition that defines among the PCT/US2008/065417, and it incorporates this paper into by carrying stating.For example, described cellulolytic enzyme prepared product comprises the polypeptide with cellulolytic enhancing activity, the polypeptide of the preferred GH61A of family, disclosed polypeptide among the preferred WO 2005/074656 (Novozymes).Described cellulolytic enzyme prepared product also can comprise beta-glucosidase enzyme, for example derive from the beta-glucosidase enzyme of Trichoderma, Aspergillus or Penicillium (Penicillium) bacterial strain, comprise disclosed fusion rotein among the WO 2008/057637 with beta-glucosidase activity.Described cellulolytic enzyme prepared product also can comprise CBH II enzyme, preferred autochthonal shuttle spore mould (Thielavia terrestris) cellobiohydrolase II CEL6A.Described cellulolytic enzyme prepared product also can comprise cellulolytic enzyme, preferably derives from the cellulolytic enzyme of Trichodermareesei or special humicola lanuginosa.
Described cellulolytic enzyme prepared product also can comprise the polypeptide with cellulolytic enhancing activity (GH61A) that is disclosed among the WO2005/074656; Beta-glucosidase enzyme (disclosed fusion rotein among the WO/2008057637) and the cellulolytic enzyme that derives from Trichodermareesei.
Cellulolytic enzyme can be commercially available product
1.5L or CELLUZYME
TM(can derive from Novozymes A/S, Denmark) or ACCELERASE
TM1000 (from Genencor Inc.USA).
Can add cellulolytic enzyme for hydrolysis through pretreated biomass slurry.Cellulolytic enzyme can be with the every gram total solids of 0.1-100FPU (TS), and the dosage in the every gram of the preferred every gram of 0.5-50FPU TS, the particularly 1-20FPU TS scope adds.In another embodiment, will be at least the every gram total solids of 0.1mg cellulolytic enzyme (TS), the preferred every gram of 3mg cellulolytic enzyme TS at least, TS is used for hydrolysis as the every gram of 5-10mg cellulolytic enzyme.
Endoglucanase (EG)
In hydrolytic process, can there be one or more endoglucanase.Term " endoglucanase " mean in-1,4-(1,3; 1,4)-callose-4-glucan hydrolase (E.C.No.3.2.1.4), in its catalyse cellulose, derivatived cellulose (as carboxymethyl cellulose and Natvosol), the moss starch 1,4-β-D-glycosidic link, mixed type β-1,3-dextran such as cereal callose or xyloglucan, and other contain β-1 in the vegetable material of cellulose components, the interior hydrolysis of 4-key.Endoglucanase activity can use carboxymethyl cellulose (CMC) hydrolysis according to Ghose, and 1987, the method for Pure and Appl.Chem.59:257-268 is determined.
Endoglucanase can derive from the bacterial strain of Trichoderma, the bacterial strain of preferred Trichodermareesei; The bacterial strain of Humicola is as the bacterial strain of special humicola lanuginosa; Or the bacterial strain of Chrysosporium, the bacterial strain of preferred Chrysosporium lucknowense.
Cellobiohydrolase (CBH)
In hydrolytic process, can there be one or more cellobiohydrolases.Term " cellobiohydrolase " means 1,4-callose cellobiohydrolase (E.C.3.2.1.91), it is at Mierocrystalline cellulose, cell-oligosaccharide or any β-1 that contains, catalysis 1 in the polymkeric substance of the glucose that 4-connects, the hydrolysis of 4-β-D-glucoside bond is from the reduction or the non-reduced terminal cellobiose that discharges of chain.
The example of cellobiohydrolase is above mentioned, comprises CBH I and CBH II from Trichodermareesei, special humicola lanuginosa; With from the mould CBH II cellobiohydrolase (CELL6A) of autochthonal shuttle spore.
Cellobiohydrolase activity can be according to by Lever etc., and 1972, Anal.Biochem.47:273-279 and by van Tilbeurgh etc., 1982, FEBSLetters 149:152-156; Van Tilbeurgh and Claeyssens, 1985, the method that FEBS Letters 187:283-288 describes is determined.The method of described Lever etc. is applicable to cellulosic hydrolysis in the assessment corn stalk, and the method for van Tilbeurgh etc. is applicable to the activity of determining cellobiohydrolase based on fluorescence two sugar derivativess.
Beta-glucosidase enzyme
In hydrolytic process, can there be one or more beta-glucosidase enzymes.Term " beta-glucosidase enzyme " means β-D-glucoside glucose hydrolysis enzyme (E.C.3.2.1.21), the hydrolysis of the terminal irreducibility β of its catalysis-D-glucosyl residue, and discharge β-D-glucose.For the present invention, beta-glucosidase activity is according to by Venturi etc., and 2002, the basic skills that J.Basic Microbiol.42:55-66 describes is determined, is the different condition of use as described herein.The beta-glucosidase activity of one unit is defined as at 100mM Trisodium Citrate, 0.01%
Produce 1.0 micromolar p-NPs at 50 ℃, pH 5 from 4mM p-nitrophenyl-β-D-glycopyranoside per minute in 20 as substrate.
Described beta-glucosidase enzyme can be originated from fungus, for example the bacterial strain of Trichoderma, Aspergillus or Penicillium.Described beta-glucosidase enzyme can derive from Trichodermareesei, as the beta-glucosidase enzyme (referring to Fig. 1 of EP 562003) by the bgl1 genes encoding.Described beta-glucosidase enzyme can derive from aspergillus oryzae (according to WO 2002/095014 generation of recombinating) in aspergillus oryzae, Aspergillus fumigatus (Aspergillus fumigatus) (according to the embodiment 22 of WO 2002/095014 generation of in aspergillus oryzae, recombinating), or aspergillus niger (1981, J.Appl. the 3rd roll up, pp157-163).
Hemicellulase
Hemicellulose can by seminase and/or acid hydrolysis decompose with discharge its five and the hexose component.Ligno-cellulose deutero-material can be handled with one or more hemicellulases.Can use any hemicellulase that is applicable to hydrolyzed hemicellulose (selective hydrolysis is wood sugar).
Preferred hemicellulase comprises zytase, arabinofuranosidase, acetyl xylan esterase, feruloyl esterase, glucuronidase, inscribe Galactanase, mannase, inscribe or circumscribed arabinase, circumscribed Galactanase and above-mentioned two or more mixture.Preferably, be used for hemicellulase of the present invention (exo-acting) hemicellulase for outer effect, and more preferably, described hemicellulase is the hemicellulase of following outer effect, it has at pH below 7, the ability of hydrolyzed hemicellulose under the acidic conditions of preferred 3-7.The example that is applicable to hemicellulase of the present invention comprises VISCOZYME
TM(can be from Novozymes A/S, Denmark obtains).
Described hemicellulase can be zytase.Described zytase can be preferably microbe-derived, as (for example, Trichoderma, Polyporus (Meripilus), Humicola, Aspergillus, the fusarium (Fusarium)) of originated from fungus or from bacterium (for example, bacillus (Bacillus)).Described zytase can derive from filamentous fungus, preferably derives from Aspergillus, as the bacterial strain of microorganism Aspergillus aculeatus (Aspergillus aculeatus), or Humicola, the preferably bacterial strain of thin cotton shape humicola lanuginosa (Humicola lanuginosa).In described zytase can be preferably-1, the 4-beta-xylanase, more preferably GH10 or GH11's is interior-1, the 4-beta-xylanase.The example of commercial xylanase comprises the A/S from Novozymes, the SHEARZYME of Denmark
TMAnd BIOFEEDWHEAT
TM
Described hemicellulase is the effectively amount of hydrolyzed hemicellulose interpolation also, as, arrive 0.5wt% total solids (TS), more preferably from about 0.05 to 0.5wt%TS amount interpolation with about 0.001.
Zytase also can 0.001-1.0g/kg dry-matter (DM) substrate amount, preferably with the amount of 0.005-0.5g/kg DM substrate, and most preferably add with the amount of 0.05-0.10g/kg DM substrate.
Xylose isomerase
Xylose isomerase (D-wood sugar ketone isomerase) (E.C 5.3.1.5) becomes the enzyme of the reversible isomerization reaction of D-xylulose for catalysis D-wood sugar.Glucose isomerase enzymatic conversion D-glucose is to the reversible isomerization of D-fructose.Yet, be called xylose isomerase during glucose isomerase.
Xylose isomerase can be used for the inventive method or technology, and can be and anyly have the active enzyme of xylose isomerase, and can derive from any source, and preferred bacterium or originated from fungus are as filamentous fungus or yeast.The example of bacterium xylose isomerase comprises those that belong to streptomyces (Streptomyces), actinoplanes (Actinoplanes), bacillus and Flavobacterium (Flavobacterium), and the thermobacillus genus (Thermotoga) of dwelling, for example new Apollo thermobacillus (the T.neapolitana) (Vieille etc. of dwelling, 1995, Appl.Environ.Microbiol.61 (5), 1867-1875) and Thermotoga maritima (T.maritime).The example of fungi xylose isomerase is the bacterial classification in Basidiomycetes (Basidiomycetes) source.
Preferred xylose isomerase derives from the bacterial strain of yeast mycocandida, preferred Candida boidinii (Candida boidinii), particularly by for example Vongsuvanlert etc., 1988, Agric.Biol.Chem., 52 (7): the disclosed Candida boidinii xylose isomerase of 1817-1824.Described xylose isomerase can preferably derive from the bacterial strain (Kloeckera 2201) of Candida boidinii, it is with DSM 70034 and ATCC 48180 preservations, be disclosed in Ogata etc., Agric.Biol.Chem, 33,1519-1520 or Vongsuvanlert etc., 1988, Agric.Biol.Chem, 52 (2), 1519-1520.
In one embodiment, described xylose isomerase derives from the bacterial strain of streptomyces, for example, derive from the bacterial strain (U.S. Patent number 4 of mouse ash streptomycete (Streptomyces murinus), 687,742), streptomyces flavovirens (S.flavovirens), streptomyces albus (S.albus), do not produce look streptomycete (S.achromogenus), thorniness streptomycete (S.echinatus), Vad More streptomycete (S.wedmorensis), it is disclosed in United States Patent (USP) 3,616, No. 221.Other xylose isomerase is disclosed in United States Patent (USP) 3,622, and No. 463, United States Patent (USP) 4,351, No. 903, United States Patent (USP) 4,137, No. 126, United States Patent (USP) 3,625, No. 828, No. 12,415, HU patent, DE patent 2,417,642, JP patent 69,28, No. 473, and WO 2004/044129, each all incorporates this paper into by carrying stating.Described xylose isomerase can be immobilization or liquid form.The preferred liquid form.The example of commercial available xylose isomerase comprises the A/S from Novozymes, the SWEETZYME of Denmark
TMT.The amount of the xylose isomerase that adds provides the activity level in the every gram total solids of the 0.01-100IGIU scope.
α-Dian Fenmei
Can use one or more α-Dian Fenmei.Preferred α-Dian Fenmei is microbe-derived, as bacterium or originated from fungus.Optimal α-Dian Fenmei is based on that processing condition determine, but those skilled in the art can easily determine.
Preferred α-Dian Fenmei is an acid alpha-amylase, for example, and fungi acid alpha-amylase or bacterium acid alpha-amylase.Phrase " acid alpha-amylase " means α-Dian Fenmei (E.C.3.2.1.1), and is preferred 3.5 to 63 to 7 when it adds with significant quantity, or more preferably the pH in the scope of 4-5 has optimum activity.
Bacterial
As implied above, described α-Dian Fenmei can be the bacillus source.Described bacillus α-Dian Fenmei can preferably derive from Bacillus licheniformis (Bacillus licheniformis), bacillus amyloliquefaciens (Bacillus amyloliquefaciens), the bacterial strain of subtilis (Bacillus subtilis) or bacstearothermophilus (Bacillus stearothermophilus), but also can derive from other bacillus bacterial classifications.The particular instance of the α-Dian Fenmei that contains comprises the bacillus licheniformis alpha-amylase of the SEQ ID NO:4 that is shown in WO 1999/19467, be shown in WO 1999/19467 SEQ ID NO:5 the bacillus amyloliquefaciens α-Dian Fenmei and be shown in the bacstearothermophilus α-Dian Fenmei (all sequences is incorporated this paper into by carrying stating) of the SEQ ID NO:3 of WO 1999/19467.In one embodiment, described α-Dian Fenmei can be and is shown in the WO 1999/19467 SEQ ID NO:1 of (incorporating this paper into by carrying stating) respectively, any sequence in 2 or 3 has at least 60%, preferably at least 70%, more preferably at least 80%, even more preferably at least 90%, for example at least 95%, the enzyme of at least 96%, at least 97%, at least 98% or at least 99% identity degree.
Described bacillus α-Dian Fenmei also can be variant and/or heterozygote, particularly is described in arbitrary variant and/or heterozygote among WO1996/23873, WO 1996/23874, WO 1997/41213, WO 1999/19467, WO2000/60059 and the WO 2002/10355 (All Files is incorporated this paper into by carrying stating).The alpha-amylase variants of containing especially is disclosed in United States Patent (USP) 6,093,562,6,297,038 or 6,187, No. 576 (incorporating this paper into) by carrying stating, and be included in position R179 has one or two aminoacid deletion to G182 bacstearothermophilus α-Dian Fenmei (BSG α-Dian Fenmei) variant, preferred WO 1996/023873 disclosed two disappearances-referring to, for example, the 20th page of 1-10 capable (incorporating this paper into) by carrying stating, preferably compare corresponding to Δ (181-182), or use numbering disappearance amino acid R179 and the G180 of the SEQID NO:3 among the WO 1999/19467 with the listed wild-type BSG α-Dian Fenmei aminoacid sequence of WO 1999/19467 disclosed SEQ ID NO:3.Even bacillus α-Dian Fenmei more preferably, bacstearothermophilus α-Dian Fenmei particularly, it has two disappearances corresponding to Δ (181-182) than the listed wild-type BSG α-Dian Fenmei aminoacid sequence of WO 99/19467 disclosed SEQ ID NO:3, and comprises that further N193F replaces (also being expressed as I181*+G182*+N193F).
Bacterium heterozygote α-Dian Fenmei
Can use one or more bacterium heterozygote α-Dian Fenmei.The heterozygote α-Dian Fenmei that contains especially comprises 445 C-terminal amino acid residues of bacillus licheniformis alpha-amylase (being shown in the SEQ ID NO:4 of WO 99/19467), and 37-terminal amino acid residues that derive from the α-Dian Fenmei (being shown in the SEQ ID NO:5 of WO99/19467) of bacillus amyloliquefaciens, and have one or more, particularly whole in the following replacement:
48A+T49I+G107A+H156Y+A181T+N190F+I201F+A209V+Q264S (using the Bacillus licheniformis numbering of the SEQ ID NO:4 of WO 99/19467).The variant (or the sudden change of the correspondence in other bacillus α-Dian Fenmei skeletons) that also preferably has one or more following sudden changes: H154Y, A181T, N190F, the disappearance of two residues between A209V and Q264S and/or position 176 and 179, the disappearance of preferred E178 and G179 (using the SEQ ID NO:5 numbering of WO 99/19467).
Fungal alpha-amylase
Can use one or more fungal alpha-amylases.Fungal alpha-amylase comprises the α-Dian Fenmei that derives from the Aspergillus bacterial strain, as aspergillus oryzae (Aspergillus oryzae), and aspergillus niger (Aspergillus niger) and valley aspergillus (Aspergillis kawachii) α-Dian Fenmei.
Preferred acid fungal alpha-amylase is a Fungamyl sample α-Dian Fenmei, and it derives from the bacterial strain of aspergillus oryzae.Phrase " Fungamyl sample α-Dian Fenmei " refers to following α-Dian Fenmei, and promptly the maturing part with aminoacid sequence shown in the SEQ ID NO:10 of WO 1996/23874 shows high identity, promptly, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 95%, more than 96%, more than 97%, more than 98%, more than 99% or even 100% identity.
Another preferred acid alpha-amylase derives from the bacterial strain of aspergillus niger.Described acid fungal alpha-amylase can be the α-Dian Fenmei from aspergillus niger, and it is disclosed in the Swiss-prot/TeEMBL database with original accession number P56271 as " AMYA_ASPNG ", and is described in WO 1989/01969 (embodiment 3).The commercial available acid fungal alpha-amylase that derives from aspergillus niger is SP288 (can be by Novozymes A/S, Denmark obtains).
Described fungal alpha-amylase also can be the wild-type enzyme (being non-heterozygote) that comprises starch binding domain (SBD) and α-Dian Fenmei catalytic domain, or its variant.In one embodiment, described wild-type α-Dian Fenmei can derive from the bacterial strain of valley aspergillus (Aspergillus kawachii).
Other wild-type α-Dian Fenmei that contain comprise the bacterial strain that derives from Rhizomucor (Rhizomucor) and Polyporus (Meripilus), those α-Dian Fenmei of preferred Rhizomucor pusillus (Rhizomucor pusillus) (WO 2004/055178 incorporates this paper into by carrying stating) or huge pore fungus (Meripilus giganteus) bacterial strain.
α-Dian Fenmei can derive from as Kaneko etc. 1996, J.Ferment.Bioeng.81:292-298 " Molecular-cloning and determination of the nucleotide-sequence of a gene encoding an acid-stable α-amylase from Aspergillus kawachii " is open, and is further used as the disclosed valley aspergillus of EMBL:#AB008370.
Fungi heterozygote α-Dian Fenmei
Can use one or more fungi heterozygote α-Dian Fenmei.Described fungi acid alpha-amylase can be the heterozygote α-Dian Fenmei.The example of fungi heterozygote α-Dian Fenmei comprises that being disclosed in WO 2005/003311 or U. S. application discloses in No. 2005/0054071 (Novozymes) or the U.S. Patent application 60/638, No. 614 (Novozymes) those, incorporates it into this paper by carrying stating.The heterozygote α-Dian Fenmei can comprise α-Dian Fenmei catalytic domain (CD) and sugared in conjunction with territory/module (CBM), as starch binding domain, and optional joint.
The particular instance of the heterozygote α-Dian Fenmei that is contained comprises Application No. 60/638, table 1 among 614 embodiment those disclosed in 5, comprise Fungamyl variant (US application 60/638 with catalytic domain JA118 and Luo Eratai bacterium (Athelia rolfsii) SBD, SEQ ID NO:100 in No. 614), Rhizomucor pusillus α-Dian Fenmei (US application 60/638 with Luo Eratai bacterium AMG joint and SBD, SEQ ID NO:101 in No. 614), (it is as U. S. application number 11/316 to have the Rhizomucor pusillus α-Dian Fenmei of aspergillus niger glucoamylase joint and SBD, aminoacid sequence SEQ ID NO:20 in 535, the combination of SEQ IDNO:72 and SEQ ID NO:96 is disclosed in table 5), or as the V039 in the table 5 among the WO2006/069290, with the huge pore fungus α-Dian Fenmei with Luo Eratai bacterium glucoamylase joint and SBD (the SEQ ID NO:102 in the US application 60/638,614).Other heterozygote α-Dian Fenmei that contain especially are listed any heterozygote α-Dian Fenmei in the table 3,4,5 and 6 among U. S. application number 11/316,535 and WO 2006/069290 (each all incorporates this paper into by the carrying stating) embodiment 4.
Other particular instances of the heterozygote α-Dian Fenmei that contains comprise that U. S. application discloses those disclosed in No. 2005/0054071, comprise the 15th page table 3 those disclosed, as have the aspergillus niger α-Dian Fenmei of valley aspergillus joint and starch binding domain.
Also contain following α-Dian Fenmei, itself and any above mentioned α-Dian Fenmei show high identity, promptly, show more than 70% with the maturing enzyme sequence, more than 75%, more than 80%, more than 85%, more than 90%, more than 95%, more than 96%, more than 97%, more than 98%, more than 99% or even 100% identity.
Acid alpha-amylase can be according to the present invention with 0.1 to 10AFAU/g DS, and preferred 0.10 to 5AFAU/g DS, the particularly amount of 0.3 to 2AFAU/g DS add.
Commercial α-Dian Fenmei product
The commercial composition that preferably comprises α-Dian Fenmei comprises the MYCOLASE from DSM; BAN
TM, TERMAMYL
TMSC, FUNGAMYL
TM, LIQUOZYME
TMX and SAN
TMSUPER, SAN
TMEXTRA L (Novozymes A/S) and CLARASE
TML-40,000, DEX-LO
TM, SPEZYME
TMFRED, SPEZYME
TMAA and SPEZYME
TMDELTAAA (Genencor Int.), and the acid fungal alpha-amylase of selling with trade(brand)name SP288 (can be by Novozymes A/S, Denmark obtains).
The sugar source generates enzyme
Phrase " sugared source generation enzyme " comprises glucoamylase (it is glucose generation person), beta-amylase and product maltogenic amylase (it is maltose generation person).The sugar source generates endonuclease capable and produces sugar, and it can be by described fermenting organism as energy source, for example, and when being used to produce tunning for example during alcoholic acid technology.The sugar that is produced can be direct or indirect be converted into desired fermentation product, preferred alcohol.Can exist sugared source to generate the mixture of enzyme.The mixture of containing especially is for being glucoamylase and α-Dian Fenmei at least, acid starch enzyme particularly, even the more preferably mixture of acid fungal alpha-amylase.
Glucoamylase
Can use one or more glucoamylases.Glucoamylase can derive from any suitable source, for example derives from microorganism or plant.Preferred glucoamylase is fungi or bacterial origin, be selected from down group: the Aspergillus glucoamylase, particularly aspergillus niger G1 or G2 glucoamylase (Boel etc., 1984, EMBO is (5) J.3: p.1097-1102), and variant, as be disclosed in WO 1992/00381, WO2000/04136 and WO 2001/04273 (from Novozymes, Denmark) those; Be disclosed in Aspergillus awamori (A.awamori) glucoamylase of WO 1984/02921, the aspergillus oryzae glucoamylase (Agric.Biol.Chem., 1991,55 (4): p.941-949), and variant or fragment.Other Aspergillus glucoamylase variants comprise the variant with enhanced thermostability: G137A and G139A (Chen etc., 1996, Prot.Eng.9:499-505); D257E and D293E/Q (Chen etc., 1995, Prot.Eng.8,575-582); N182 (Chen etc., 1994, Biochem.J.301:275-281); Disulfide linkage, A246C (Fierobe etc., 1996, Biochemistry, 35:8698-8704); And at A435 and S436 position importing Pro residue (Li etc., 1997, Protein Eng.10:1199-1204).
Other glucoamylase comprises that Luo Eratai bacterium (before being expressed as sieve ear photovoltaicing leather bacteria (Corticium rolfsii)) glucoamylase is (referring to United States Patent (USP) 4,727, No. 026 and Nagasaka etc., 1998, " Purification and properties of the raw-starch-degrading glucoamylases from Corticium rolfsii; Appl Microbiol Biotechnol.50:323-330); and Talaromyces (Talaromyces) glucoamylase; particularly derive from Ai Mosen ankle joint bacterium (Talaromyces emersonii) (WO 1999/28448); Talaromyces leycettanus (United States Patent (USP) Re.32; No. 153), Du Pont ankle joint bacterium (Talaromyces duponti) and thermophilic ankle joint bacterium (Talaromyces thermophilus) (United States Patent (USP) 4,587, No. 215).
The bacterium glucoamylase of containing comprises from fusobacterium, particularly (EP 135 for pyrolysis clostridium amylobacter (C.thermoamylolyticum), 138) and the glucoamylase of hot sulfurization hydrogen clostridium (C.thermohydrosulfuricum) (WO 1986/01831) and lobe ring bolt bacterium (Trametes cingulata), the latter is disclosed in WO 2006/069289 (incorporating this paper into by carrying stating).
Also contained the heterozygote glucoamylase.The example of described heterozygote glucoamylase is disclosed in WO2005/045018.Specific examples comprises the table 1 that is disclosed in WO 2005/045018 embodiment 1 and 4 heterozygote glucoamylase, and its degree with its instruction heterozygote glucoamylase is incorporated this paper into by carrying stating.
Also contained the glucoamylase that shows high identity with any above mentioned glucoamylase, that is, show more than 70%, more than 75% with the maturing enzyme sequence, more than 80%, more than 85%, more than 90%, more than 95%, more than 96%, more than 97%, more than 98%, more than 99% or even 100% identity.
The commercial available composition that comprises glucoamylase comprises AMG 200L, AMG 300L, SAN
TMSUPER, SAN
TMEXTRA L, SPIRIZYME
TMPLUS, SPIRIZYME
TMFUEL, SPIRIZYME
TMB4U and AMG
TME (from Novozymes A/S); OPTIDEX
TM300 (from Genencor Int.); AMIGASE
TMAnd AMIGASE
TMPLUS (from DSM); G-ZYME
TMG900, G-ZYME
TMAnd G990ZR (from Genencor Int.).
Glucoamylase can be with 0.02-20AGU/g DS, and preferred 0.1-10AGU/g DS particularly at 1-5AGU/g DS, adds as the amount of 0.5AGU/g DS.
Beta-amylase
Can use one or more beta-amylases.The title that term " beta-amylase " (E.C 3.2.1.2) produces maltogenic amylase for (exo-acting) that gives outer effect traditionally, in its catalysis amylose starch, amylopectin and the relevant glucose polymer 1, the hydrolysis of 4-α-glucoside bond.Remove the maltose unit continuously until molecular degradation from non-reducing chain end in progressively mode, perhaps, under the situation of amylopectin, until arriving branching-point.The maltose that discharges has β anomer conformation, obtains the title of beta-amylase thus.
From various plants and microorganism, separated beta-amylase (W.M.Fogarty and C.T.Kelly, Progress in Industrial Microbiology, the 15th volume, pp.112-115,1979).These beta-amylases be characterised in that have scope in 40 ℃ to 65 ℃ optimum temperuture and scope at 4.5 to 7 optimal pH.Commercial available beta-amylase from barley is from Novozymes A/S, the NOVOZYM of Denmark
TMWBA and from Genencor Int., the SPEZYME of USA
TMBBA 1500.
Produce maltogenic amylase
Can use one or more to produce maltogenic amylase.Amylase also can be the product maltogenic alpha-amylase enzyme.(dextran 1,4-α-maltose lytic enzyme E.C.3.2.1.133) can be hydrolyzed into amylose starch and amylopectin the maltose of α-conformation to produce maltogenic alpha-amylase enzyme.Can obtain by Novozymes A/S from the product maltogenic amylase of bacstearothermophilus bacterial strain NCIB11837 is commercial.The α-Dian Fenmei of producing maltose is described in United States Patent (USP) 4,598, and 048,4,604,355 and 6,162, No. 628, it incorporates this paper into by carrying stating.Described product maltogenic amylase can 0.05-5mg total protein/gram DS or the amount of 0.05-5MANU/g DS add.
Proteolytic enzyme
Proteolytic enzyme can add in hydrolysis, fermentation or hydrolysis simultaneously and fermenting process.Can add proteolytic enzyme during the fermentation with deflocculated fermenting organism, particularly yeast.Described proteolytic enzyme can be any proteolytic enzyme.In a preferred embodiment, described proteolytic enzyme is microbe-derived aspartic protease, preferred fungi or bacterial origin.Acid fungal protease is preferred, but also can use other proteolytic enzyme.
Suitable proteolytic enzyme comprises microbial protease, for example fungi and bacteria protease.Preferred proteolytic enzyme is aspartic protease, that is, be characterized as can be under the acidic conditions below the pH7 proteolytic enzyme of protein hydrolysate.
The acid fungal protease of containing comprises and derives from Aspergillus, and Mucor (Mucor), Rhizopus (Rhizopus), mycocandida, Coriolus Qu61 (Coriolus), inner seat shell belong to the fungal proteinase that (Endothia), entomophthora belong to (Enthomophtra), rake teeth Pseudomonas (Irpex), Penicillium (Penicillium), Rhizoctonia (Sclerotium) and torulopsis (Torulopsis).Contain especially be derive from aspergillus niger (referring to, for example, Koaze etc., 1964, Agr.Biol.Chem.Japan, 28,216), saitox aspergillus (Aspergillus saitoi) (referring to, for example, Yoshida, 1954, J.Agr.Chem.Soc.Japan, 28,66), Aspergillus awamori (Hayashida etc., 1977Agric.Biol.Chem., 42 (5), 927-933), the proteolytic enzyme of microorganism Aspergillus aculeatus (WO1995/02044) or aspergillus oryzae, as pepA proteolytic enzyme, and from Mucor pusillus (Mucor pusillus) and rice black wool mould (Mucor miehei) aspartic protease.
Also contain neutrality or Sumizyme MP, as derived from the proteolytic enzyme of Bacillus strain.For example, the proteolytic enzyme that the present invention is contained derives from bacillus amyloliquefaciens, and has
Can be used as login at Swissprot Number P06832The sequence that obtains.Also contain with
Can be used as accession number P06832 at SwissprotThe aminoacid sequence that obtains has at least 90% identity, as at least 92%, and at least 95%, at least 96%, at least 97%, at least 98% or be in particular the proteolytic enzyme of at least 99% identity.
Further contain be with WO 2003/048353 in have at least 90% identity as the disclosed aminoacid sequence of SEQ ID NO:1, as at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or be in particular the proteolytic enzyme of at least 99% identity.
Also contain papoid sample proteolytic enzyme, as the proteolytic enzyme in the E.C.3.4.22.* (L-Cysteine HCL Anhydrous), as EC 3.4.22.2 (papoid), EC 3.4.22.6 (Disken), EC 3.4.22.7 (asclepain (asclepain)), EC 3.4.22.14 (Actinidin (actinidain)), EC3.4.22.15 (cathepsin L), EC 3.4.22.25 (glycyl endopeptidase) and EC 3.4.22.30 (caricin (caricain)).
In one embodiment, described proteolytic enzyme can be and derives from Aspergillus, as the protease preparation of the bacterial strain of aspergillus oryzae.In another embodiment, described proteolytic enzyme can derive from Rhizomucor, the bacterial strain of preferred Man Hegen Mucor (Rhizomucor miehei).In the embodiment that another is contained, described proteolytic enzyme can be protease preparation, preferably derives from the proteolysis prepared product of bacterial strain of Aspergillus (as aspergillus oryzae) and the mixture of proteolytic enzyme that derives from the bacterial strain of Rhizomucor (preferred Man Hegen Mucor).
Aspartate protease is described in, for example, Handbook of Proteolytic Enzymes, A.J.Barrett, N.D.Rawlings and J.F.Woessner compile, Academic Press, San Diego, 1998,270 chapters).The suitable example of aspartate protease comprises, for example, and R.M.Berka etc., Gene, 96,313) (1990); (Gene such as R.M.Berka, 125,195-198) (1993); And Gomi etc., Biosci.Biotech.Biochem.57,1095-1100 (1993) those disclosed, it incorporates this paper into by carrying stating.
Commercial available product comprises
ESPERASE
TM, FLAVOURZYME
TM, PROMIX
TM,
NOVOZYM
TMFM 2.0L and NOVOZYM
TM50006 (can obtain) and from Genencor Int. by Novozymes A/S, Denmark, the GC106 of Inc.USA.
TMAnd SPEZYME
TMFAN.
Described proteolytic enzyme can the every gram of 0.0001-1mg zymoprotein DS, and preferred 0.001 amount to the every gram of 0.1mg zymoprotein DS exists.Perhaps, described proteolytic enzyme can 0.0001 to 1LAPU/g DS, preferred 0.001 to 0.1LAPU/g DS and/or 0.0001 to 1mAU-RH/g DS, preferred 0.001 amount that arrives 0.1mAU-RH/g DS exist.
Describe also claimed invention herein and be not limited to the scope of disclosed specific embodiments herein, because these embodiments are intended to illustrate several aspect of the present invention.Intention comprises the embodiment of any equivalence and the combination of one or more described embodiments within the scope of the invention.According to the description of preamble, illustrate herein and the modification put down in writing outside can be conspicuous to various modifications of the present invention for the undergraduate course those skilled in the art.Be intended to make these modifications also to fall in the scope of claims.
Quoted many pieces of reference herein, their integral body has been incorporated into by carrying stating.Further describe the present invention by following examples, but these embodiment should be interpreted as limitation of the scope of the invention.
Material and method
Method:
Identity
Dependency between two aminoacid sequences or two nucleotide sequences is described by parameter " identity ".
For the present invention, the identity degree between two aminoacid sequences can be passed through Clustal method (Higgins, 1989, CABIOS 5:151-153) and uses LASERGENE
TMMEGALIGN
TMSoftware (DNASTAR, Inc., Madison, WI) and identity table and following multiple ratio (multiple alignment) parameter is determined: the breach point penalty is 10, and the notch length point penalty is 10.Pairing comparison parameter (pairwise alignment parameter) is K tuple (Ktuple)=1, breach point penalty=3, window=5 and diagonal lines=5.
For the present invention, the identity between two nucleotide sequences can be passed through Wilbur-Lipman method (Wilbur and Lipman, 1983, Proceedings of the National Academy of Science USA80:726-730) and uses LASERGENE
TMMEGALIGN
TMSoftware (DNASTAR, Inc., Madison, WI) and identity table and following multiple ratio parameter is determined: the breach point penalty is 10, and the notch length point penalty is 10.Pairing comparison parameter is K tuple=3, breach point penalty=3 and window=20.
Glucoamylase activity (AGU)
Novo glucose starch unit of enzyme (AGU) is defined as at 37 ℃, pH4.3, substrate: maltose 23.2mM, damping fluid: acetate 0.1M, the enzyme amount of per minute hydrolysis 1 micromole's maltose under the standard conditions in 5 minutes reaction times.
Can use the automatic analyser system.Mutarotase (mutarotase) is added in the Hexose phosphate dehydrogenase reagent, make any alpha-D-glucose that exists be converted into β-D-glucose.Hexose phosphate dehydrogenase reacts in above-mentioned reaction with β-D-glucose specifically, forms NADH, and it uses photometer to measure measuring as initial glucose concn at the 340nm place.
| The AMG incubation: | |
| Substrate: | Maltose 23.2mM |
| Damping fluid: | Acetate 0.1M |
| pH： | 4.30±0.05 |
| Heated culture temperature: | 37℃±1 |
| Reaction times: | 5 minutes |
| The enzyme working range: | 0.5-4.0AGU/mL |
| Color reaction: | |
| GlucDH： | 430U/L |
| Mutarotase: | 9U/L |
| NAD： | 0.21mM |
| Damping fluid: | Phosphoric acid salt 0.12M; 0.15MNaCl |
| pH： | 7.60±0.05 |
| Heated culture temperature | 37℃±1 |
| Reaction times: | 5 minutes |
| Wavelength: | 340nm |
The folder of this analytical procedure of more detailed description (EB-SM-0131.02/01) can be as requested by Novozymes A/S, and Denmark obtains, and it incorporates this paper into by carrying stating.
Alpha-amylase activity (KNU)
Alpha-amylase activity can use yam starch to determine as substrate.This method is based on the decomposition of enzyme for modified potato starch, and mixes with iodine solution by the sample with starch/enzyme solution and to follow the tracks of reaction.Originally, formed black-and-blue (blackish blue), but in the amylolysis process, blueness is more and more lighter, and gradually becomes reddish-brown (reddish-brown), itself and tinted shade standard (colored glass standard) are compared.
One thousand Novo α-Dian Fenmei unit (KNU) is defined as under standard conditions (that is, 37 ℃+/-0.05; 0.0003M Ca
2+And pH 5.6) the required enzyme amount of starch dry matter Merck Amylum Solubile of dextrinization 5260mg.
The folder EB-SM-0009.02/01 of this analytical procedure of more detailed description can be as requested by Novozymes A/S, and Denmark obtains, and it incorporates this paper into by carrying stating.
Acid alpha-amylase activity (AFAU)
When used according to the invention, the activity of acid alpha-amylase can be measured with FAU-F (fungal alpha-amylase unit) or AFAU (acid fungal alpha-amylase unit).
Determine FAU-F
FAU-F fungal alpha-amylase unit (Fungamyl) measures with respect to the enzyme standard substance of concentration known.
The folder of this standard method of more detailed description (EB-SM-0216.02) can be as requested by Novozymes A/S, and Denmark obtains, and incorporates this document folder into this paper by carrying stating.
Acid alpha-amylase activity (AFAU)
The acid alpha-amylase activity can be measured by AFAU (acid fungal alpha-amylase unit), and it is determined with respect to the enzyme standard substance.1AFAU is defined as the enzyme amount of the 5.260mg starch dry matter of per hour degrading under the standard conditions of mentioning below.
Acid alpha-amylase, its be the inscribe α-Dian Fenmei (1,4-α-D-dextran-glucan hydrolase, the E.C.3.2.1.1) α-1 in the hydrolyzed starch intramolecule zone, 4-glucoside bond have the oligosaccharides and the dextrin of different chain length with formation.The intensity of the color that forms with iodine is directly proportional with starch concentration.Use reverse colorimetry (reverse colorimetry) under the analysis condition of regulation, to measure the reduction of starch concentration as amylase activity.
α-Dian Fenmei
Starch+iodine → dextrin+oligosaccharides
λ＝590nm 40℃，pH?2.5
Blueness/purple t=23 decolours second
Standard conditions/reaction conditions:
Substrate: Zulkovsky starch, approximately 0.17g/L
Damping fluid: Citrate trianion, approximately 0.03M
Iodine (I
2): 0.03g/L
CaCl
2： 1.85mM
pH： 2.50±0.05
Heated culture temperature: 40 ℃
Reaction times: 23 seconds
Wavelength: 590nm
Enzyme concn: 0.025AFAU/mL
Enzyme working range: 0.01-0.04AFAU/mL
The folder EB-SM-0259.02/01 of this analytical procedure of more detailed description can be as requested by Novozymes A/S, and Denmark obtains, and it incorporates this paper into by carrying stating.
Use filter paper assay method (FPU assay method) to measure cellulase activity
1. method is originated
1.1 present method is disclosed in Adney and Baker, 1996.Laboratory Analytical Procedure, LAP-006, the file that is entitled as " Measurement of Cellulase Activities " of National Renewable Energy Laboratory (NREL).It is based on IUPAC method (Ghose, 1987, Measurement of Cellulse Activities, Pure ﹠amp for the mensuration cellulase activity; Appl.Chem.59:257-268).
2. method
2.1 this method such as Adney and Baker, 1996, the described enforcement that sees above only is to use the absorbance after 96 orifice plates read colour developing, and is as mentioned below.
2.2 enzymatic determination pipe:
2.2.1 (rolled) filter paper bar (#1 Whatman with rolling; 1X6cm; 50mg) be added into the bottom of test tube (13X 100mm).
2.2.2 in pipe, add 1.0mL 0.05M sodium citrate buffer solution (pH 4.80).
2.2.3 the pipe that will contain filter paper and damping fluid in circulator bath 50 ℃ of (± 0.1 ℃) incubations 5 minutes.
2.2.4 behind the incubation, Xiang Guanzhong adds the enzyme diluent in the 0.5mL citrate buffer.The enzyme diluent be designed to produce a little more than with value a little less than target value 2.0mg glucose.
2.2.5 will manage contents mixed in 3 seconds by gentle vortex concussion.
2.2.6 after the vortex concussion, with pipe in circulator bath 50 ℃ of (± 0.1 ℃) incubations 60 minutes.
2.2.7 immediately pipe was taken out from water-bath behind the incubation at 60 minutes, and in each pipe, adds 3.0mL DNS reagent with termination reaction.To manage vortex and shake for 3 seconds to mix.
2.3 blank and contrast
2.3.1 prepare reagent blank by in test tube, adding the 1.5mL citrate buffer.
2.3.2 place the bottom of test tube and add the 1.5mL citrate buffer by filter paper bar and prepare substrate contrast with rolling.
2.3.3 by the 1.0mL citrate buffer is mixed the enzyme contrast for preparing every kind of enzyme diluent with the enzyme diluent that 0.5mL suits.
2.3.4 measure reagent blank, substrate contrast and enzyme contrast in the mode identical, and carry out with the enzymatic determination pipe with the enzymatic determination pipe.
2.4 glucose standard substance
2.4.1 preparation 100mL glucose liquid storage (10.0mg/mL), and freezing 5mL aliquots containig.Before use, aliquots containig is thawed also vortex concussion to mix.
2.4.2 the following diluent that in citrate buffer, prepares liquid storage:
G1=1.0mL liquid storage+0.5mL damping fluid=6.7mg/mL=3.3mg/0.5mL
G2=0.75mL liquid storage+0.75mL damping fluid=5.0mg/mL=2.5mg/0.5mL
G3=0.5mL liquid storage+1.0mL damping fluid=3.3mg/mL=1.7mg/0.5mL
G4=0.2mL liquid storage+0.8mL damping fluid=2.0mg/mL=1.0mg/0.5mL
2.4.3 prepare glucose standard QC by in the 1.0mL citrate buffer, adding every kind of diluent of 0.5mL.
2.4.4 measure glucose standard QC in the mode identical, and carry out with the enzymatic determination pipe with the enzymatic determination pipe.
2.5 colour developing
2.5.1, all pipes were boiled in water-bath 5 minutes together at 60 minutes incubations with after adding DNS.
2.5.2 after boiling, immediately they are cooled off in ice/water-bath.
2.5.3 when cooling will be managed vortex concussion momently, and allow the paper pulp sedimentation.Then by being added into 200 microlitre ddH in 96 orifice plates from 50 microlitres of each pipe
2O dilutes each pipe.Each hole is mixed, and read absorbancy at 540nm.
2.6 calculate (example provides in the NREL file)
2.6.1 by with the glucose concn (mg/0.5mL) of four kinds of standard substance (G1-G4) to A
540Draw the glucose typical curve.This is to use linear regression (Prism Software) to come match, and uses the equation of this line to determine the glucose that each enzymatic determination pipe is generated.
2.6.2 the glucose (mg/0.5mL) that drafting is generated is to the dilution curve of total enzyme, wherein Y-axis (enzyme extent of dilution) is a logarithmically calibrated scale.
2.6.3 generating the enzyme extent of dilution that just has been higher than 2.0mg glucose and generating line of picture between the extent of dilution that just has been lower than this value.Determine accurately to generate the enzyme extent of dilution of 2.0mg glucose according to this line.
2.6.4 following calculating filter paper unit/mL (FPU/mL):
FPU/mL=0.37/ generates the enzyme extent of dilution of 2.0mg glucose
Protease assay method-AU (RH)
Protein decomposing activity can be determined as substrate with the oxyphorase of sex change.In the Anson-Hemoglobin method that is used for determining protein decomposing activity, the oxyphorase of digestion sex change precipitates indigested oxyphorase then with trichoroacetic acid(TCA) (TCA).Determine the amount of TCA soluble product with phenol reagent, described phenol reagent runs into tyrosine and tryptophane presents blueness.
One Anson unit (AU-RH) is defined as under standard conditions (promptly, 25 ℃, pH 5.5 and 10 minute reaction times) so that the solvable product amount of TCA that per minute discharges runs into the initial rate digestion oxyphorase that phenol reagent provides identical color with a millinormal tyrosine.
AU (RH) method is described in EAL-SM-0350, and can require to obtain from Novozymes A/S Denmark.
Protein decomposing activity (AU)
Protein decomposing activity can be determined as substrate with the oxyphorase of sex change.In the Anson-Hemoglobin method that is used for determining protein decomposing activity, the oxyphorase of digestion sex change precipitates indigested oxyphorase then with trichoroacetic acid(TCA) (TCA).Determine the amount of TCA soluble product with phenol reagent, described phenol reagent runs into tyrosine and tryptophane presents blueness.
One Anson unit (AU) is defined as under standard conditions (promptly, 25 ℃, pH 7.5 and 10 minute reaction times) so that the solvable product amount of TCA that per minute discharges runs into the initial rate digestion oxyphorase that phenol reagent provides identical color with a millinormal tyrosine.
The file AF4/5 of this analytical procedure of more detailed description can require to obtain from Novozymes A/S Denmark, and this document is incorporated this paper into by carrying stating in full.
Protease assay method (LAPU)
The enzyme amount that 1 leucine aminopeptidase unit (LAPU) decomposes 1 μ M substrate for per minute under the following conditions: L-leucine-p-Nitroaniline of 26mM is as substrate, 0.1M Tris damping fluid (pH 8.0), 37 ℃ and 10 minute reaction times.
LAPU is described in EB-SM-0298.02/01, and it can should require to obtain from Novozymes A/S Denmark.
Determine to produce maltogenic amylase activity (MANU)
MANU (produce maltogenic amylase Novo unit,
MAltogenic
AMylase
NOvo
UNit) may be defined as at 37 ℃, pH 5.0, react 30 minutes, discharge the required enzyme amount of micromole's maltose at the concentration per minute of every ml 0.1M citrate buffer 10mg trisaccharide maltose (Sigma M 8378).
Material:
The yeast prepared product:
Can be from Red Star/Lesaffre, the RED STAR that USA obtains
TM
Cellulase prepared product A:
Mierocrystalline cellulose decomposition and combination thing, it comprises the polypeptide (GH61A) with disclosed cellulolytic enhancing activity among the WO 2005/074656; The fusion rotein of beta-glucosidase enzyme (being disclosed in WO 2008/057637)); With the cellulolytic enzyme prepared product that derives from Trichodermareesei.Cellulase prepared product A is disclosed in common unsettled International Application PCT/US2008/065417 number.
The biomass substrate:
Unwashed through pretreated maize straw (PCS): through acid catalyzed, vapor explosion, from The National Renewable Energy Laboratory, Golden, CO obtains.
Embodiment
Tested the effect that dissolved air flotation sludge is made an addition to biomass slurry.With dissolved air flotation sludge with multiple amount before hydrolysis, make an addition to through the washing through pretreated maize straw (PCS).After initial hydrolysis, measured sugared content in 72 hours.
Dissolved air flotation sludge sample is from Clinton, and the pig of NC is butchered facility and obtains.A is used for hydrolysis with the cellulase prepared product.With dissolved air flotation sludge 121 ℃ of autoclavings 20 minutes.With treated dissolved air flotation sludge be added into through washing through pretreated maize straw (PCS) slurry, and with as shown in table 1 mixing of amount of 0.10g dissolved air flotation sludge/g biomass slurry and 0.20g dissolved air flotation sludge/g biomass slurry.With mixture with cellulase prepared product A with the amount of 6.0mg albumen/g total solid 50 ℃ of hydrolysis 72 hours.In addition, will the contrast of PCS only be arranged and the contrast hydrolysis under the same conditions of dissolved air flotation sludge only be arranged.
The content of the sugar that discharges is determined by the PHBA method, and is proved conclusively by HPLC (high pressure liquid chromatography).Shown in Fig. 1 and 2 difference, treated dissolved air flotation sludge is added into enzyme hydrolysis process has increased final sugared yield and conversion of glucose per-cent.When before hydrolysis 10%w/w dissolved air flotation sludge/lignocellulose-containing materials being added into the PCS slurry, sugared yield increases to about 27.5g/L from about 23.4g/L, and sugared transformation efficiency improves to about 83% from about 69%.When before hydrolysis 20%w/w dissolved air flotation sludge/lignocellulose-containing materials being added into the PCS slurry, sugared yield increases to about 29.7g/L from about 23.4g/L, and sugared transformation efficiency improves to about 87% from about 69%.For the contrast that dissolved air flotation sludge is only arranged, in 72 hours hydrolytic process, produced the glucose that is less than 0.5g/L, therefore show that the improvement of PCS hydrolysis is attributable to interaction favourable between dissolved air flotation sludge and the PCS, promptly dissolves the air flotation sludge reduces the xylogen deleterious effect in hydrolytic process ability.
The experiment of table 1. enzymic hydrolysis is set
Claims (20)
1. be used for producing the method for tunning, comprise from lignocellulose-containing materials:
(a) the described lignocellulose-containing materials of pre-treatment;
(b) dissolved air flotation sludge is introduced through pretreated lignocellulose-containing materials;
(c) will be exposed to lytic enzyme through pretreated lignocellulose-containing materials; With
(d) ferment to produce tunning with fermenting organism.
2. the process of claim 1 wherein that described dissolved air flotation sludge introduced this lignocellulose-containing materials before lignocellulose-containing materials is exposed to lytic enzyme.
3. the process of claim 1 wherein that described dissolved air flotation sludge is to introduce this lignocellulose-containing materials when lignocellulose-containing materials is exposed to lytic enzyme.
4. each described method of claim 1-3 is wherein introduced described lignocellulose-containing materials with about 1 to the amount of about 40%w/w dissolved air flotation sludge/lignocellulose-containing materials with described dissolved air flotation sludge.
5. each described method of claim 1-4 is wherein introduced described lignocellulose-containing materials with about 5 to the amount of about 20%w/w dissolved air flotation sludge/lignocellulose-containing materials with described dissolved air flotation sludge.
6. each described method of claim 1-3, wherein said dissolved air flotation sludge comprises the refuse from the agricultural effluent treatment process.
7. the method for claim 6, wherein said dissolved air flotation sludge comprises the refuse of the waste water treatment process relevant with slaughtered animals.
8. each described method of claim 1-7, the lytic enzyme that wherein is exposed to described lignocellulose-containing materials comprises cellulase prepared product A, or its a kind of or various ingredients.
9. each described method of claim 1-8, wherein said dissolved air flotation sludge introduce described treated before pretreated lignocellulose-containing materials.
10. the method for claim 9 wherein uses the method for the combination that comprises enzyme method, by the use of thermal means, mechanical means, chemical process or these methods to handle described dissolved air flotation sludge.
11. the method for claim 9, wherein said dissolved air flotation sludge conditioning is an autoclaving.
12. each described method of claim 1-11 wherein uses sour pre-treatment to come the described lignocellulose-containing materials of pre-treatment.
13. each described method of claim 1-12, wherein said lignocellulose-containing materials is selected from down group: maize straw, corn cob, zein fiber, switchgrass, straw, rice straw, bagasse and combination thereof.
14. a method that strengthens the enzymic hydrolysis of lignocellulose-containing materials comprises:
(a) the dissolved air flotation sludge of effective xylogen blocking-up amount is introduced described lignocellulose-containing materials and
(b) described lignocellulose-containing materials is exposed to lytic enzyme.
15. the method for claim 14 was wherein introduced described lignocellulose-containing materials with described dissolved air flotation sludge before described lignocellulose-containing materials is exposed to lytic enzyme.
16. the method for claim 14 is wherein introduced described lignocellulose-containing materials with described dissolved air flotation sludge when described lignocellulose-containing materials is exposed to lytic enzyme.
17. each described method of claim 14-16, wherein said dissolved air flotation sludge is treated before introducing described lignocellulose-containing materials.
18. each described method of claim 14-17, wherein said lignocellulose-containing materials is selected from down group: maize straw, corn cob, zein fiber, switchgrass, straw, rice straw, bagasse and combination thereof.
19. the tunning that the method that a basis comprises the steps is made:
(a) the described lignocellulose-containing materials of pre-treatment;
(b) dissolved air flotation sludge is introduced through pretreated lignocellulose-containing materials;
(c) will be exposed to the lytic enzyme of significant quantity through pretreated lignocellulose-containing materials; With
(d) ferment to produce tunning with fermenting organism.
20. a mixture comprises:
(a) lignocellulose-containing materials;
(b) dissolved air flotation sludge; With
(c) lytic enzyme.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US14139408P | 2008-12-30 | 2008-12-30 | |
| US61/141,394 | 2008-12-30 | ||
| PCT/US2009/069772 WO2010078391A2 (en) | 2008-12-30 | 2009-12-30 | Improvement of enzymatic hydrolysis of pretreated lignocellulose-containing material with dissolved air flotation sludge |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN102272315A true CN102272315A (en) | 2011-12-07 |
Family
ID=42288583
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2009801534022A Pending CN102272315A (en) | 2008-12-30 | 2009-12-30 | Improvement of enzymatic hydrolysis of pretreated lignocellulose-containing material with dissolved air flotation sludge |
Country Status (4)
| Country | Link |
|---|---|
| US (1) | US20120028299A1 (en) |
| EP (1) | EP2384365A2 (en) |
| CN (1) | CN102272315A (en) |
| WO (1) | WO2010078391A2 (en) |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104805137A (en) * | 2014-01-24 | 2015-07-29 | 华东理工大学 | Method for producing gluconic acid through bioconversion of lignocellulose |
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| BR112012007928B1 (en) * | 2009-10-09 | 2022-03-15 | American Process, Inc | Biorefinery process |
| WO2012047832A2 (en) | 2010-10-07 | 2012-04-12 | Shell Oil Company | Process for the production of alcohols from biomass |
| US8609379B2 (en) | 2010-12-20 | 2013-12-17 | Shell Oil Company | Process for the production of alcohols from biomass |
| GB2503939A (en) * | 2012-07-13 | 2014-01-15 | Kind Consumer Ltd | Products derived from tobaccco biomass |
| WO2014072394A1 (en) * | 2012-11-09 | 2014-05-15 | Dsm Ip Assets B.V. | Process for enzymatic hydrolysis of lignocellulosic material and fermentation of sugars |
| WO2014072389A1 (en) * | 2012-11-09 | 2014-05-15 | Dsm Ip Assets B.V. | Process for enzymatic hydrolysis of lignocellulosic material and fermentation of sugars |
| EP2997154B1 (en) * | 2013-05-16 | 2018-07-11 | Novozymes A/S | Enhancing enzymatic hydrolysis by enzymatic preconditioning |
| WO2014191267A1 (en) * | 2013-05-28 | 2014-12-04 | Dsm Ip Assets B.V. | Process for enzymatic hydrolysis of lignocellulosic material |
| ITTO20130888A1 (en) * | 2013-10-31 | 2015-05-01 | Biochemtex Spa | PROCEDURE FOR GROWING A MICROBIAL ORGANISM |
| CN105671088B (en) * | 2014-11-20 | 2019-05-17 | 中国科学院大连化学物理研究所 | A kind of continuous conversion common fermentation method of the improved lignocellulosic of surfactant |
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- 2009-12-30 US US13/147,513 patent/US20120028299A1/en not_active Abandoned
- 2009-12-30 CN CN2009801534022A patent/CN102272315A/en active Pending
- 2009-12-30 WO PCT/US2009/069772 patent/WO2010078391A2/en active Application Filing
- 2009-12-30 EP EP09796921A patent/EP2384365A2/en not_active Withdrawn
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104805137A (en) * | 2014-01-24 | 2015-07-29 | 华东理工大学 | Method for producing gluconic acid through bioconversion of lignocellulose |
| CN104805137B (en) * | 2014-01-24 | 2018-11-23 | 华东理工大学 | A kind of method of bioconversion lignocellulosic production gluconic acid |
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| Publication number | Publication date |
|---|---|
| WO2010078391A3 (en) | 2010-09-02 |
| WO2010078391A2 (en) | 2010-07-08 |
| US20120028299A1 (en) | 2012-02-02 |
| EP2384365A2 (en) | 2011-11-09 |
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